Project 1. Mitochondrial DNA segregation and expression prelude and is essential for the selective inheritance. Although mitochondrial DNA (mtDNA) is prone to mutation and few mtDNA repair mechanisms exist, deleterious mutations are exceedingly rare. The mechanisms of how the transmission of detrimental mtDNA mutation through maternal inheritance is restricted remain debatable. We use a Drosophilamodel to dissect the mechanisms of the mtDNA selective inheritance and answer how the mechanisms operate at a molecular level. We propose a purifying selection on the organelle level based on the developmental regulation. We found the mitochondrial fission, mitochondrial activation and selective propagation, which occur as ordered events during Drosophila oogenesis in the germarium region, are indispensable for mtDNA selective inheritance. At germarium developing cyst region 2A, the mtDNA copy number per mitochondrion is reduced to 1 to prepare for effective selection on organelle level based on individual fitness. At the following region 2B, mitochondrial activity and biogenesis are boosted, which serve as a functional test. Concurrently, the mitochondrial with functional mtDNA are selectively proliferated, while the ones with deleterious mtDNA mutation are restricted. This developmental regulated process acts orderly and synergistically to secure the functional mtDNA being transmitted through Drosophila oogenesis. Project 2. The insulin signaling actives Myc to transcriptionally boost mitochondrial biogenies during oogenesis Mitochondrial genome (mtDNA) encodes key components of electron transport chain (ETC) for oxidative phosphorylation (OXPHOS), and thereby is vital for life. Mitochondria are transmitted exclusively through maternal lineage in most metazoan, which demands mothers to furnish mature oocytes with massive amount of mitochondria to power the early embryogenesis. During normal Drosophila oogenesis, mtDNA replication is tremendously stimulated at the transition stage from initial cell division to follicle formation and depends on ETC activity. These characteristics in germline are critical for limiting the transmission of severely mutated mtDNA that causes defective ETC activity. Nonetheless, the signals triggering ETC activity and mtDNA replication remain unknown. Here, we uncover that the spatial pattern of mtDNA replication and ETC activity corresponds to that of ETC gene expression. Our targeted RNAi screening reveals that insulin and JNK pathways, together with the transcription factor Myc, activate ETC biogenesis and mtDNA replication. Mechanistically, Myc is post-transcriptionally regulated by insulin signaling via its stability, both of which exhibit the same spatial pattern as mtDNA replication. A transient JNK activation at the transition of follicle formation boosts insulin signaling activity through the regulation of insulin receptor (InR) transcription. An insulin signaling-Myc feed-forward loop maintains the high insulin signaling activity in germline. Furthermore, the JNK-insulin-Myc signaling relay plays an essential physiological role for ensuring female fertility and restricting the inheritance of deleterious mtDNA variants. Altogether, our studies demonstrate a link by the developmental program that couples mitochondrial respiration to oocyte competence. Project 3. Controlled local protein synthesis limits the transmission of deleterious mitochondrial DNA mutations Damaging mtDNA mutations are effectively restricted from transmission to the next generation. This selective inheritance could result from proliferation competition, as mtDNA replication preferentially takes places in healthy organelles in the Drosophila ovary. However, it remains unknown how germ cells recognize the fitness of individual mitochondria, and boost the propagation of healthy ones selectively. Herein, we demonstrate that the outer-mitochondrial-membrane protein PINK1 selectively accumulates on mitochondria enriched in mutant genomes. PINK1 phosphorylates Larp to inhibit the local protein synthesis on the mitochondrial outer membrane that normally drives the prodigious mitochondrial biogenesis and mtDNA replication in ovary. We conclude that impaired local translation on defective mitochondria effectively limits the replication and the transmission of mtDNA mutations to the next generation. Project 4. Mitochondrial OXPHOS regulate Drosophila intestinal stem cells differentiation. Stem cells often emphasize on glycolysis for energy production, whereas the mitochondrial activation, switch from glycolysis to oxidative phosphorylation (OXPHOS), is believed to be essential for stem cell differentiation. Mitochondrial inactivation has been considered as an efficient way to facilitate the somatic cell-pluripotent stem cell reprogramming by reversing this metabolic transition. However, the cellular signaling orchestrating the metabolic shift is largely unknown. The link between mitochondrial OXPHOS and stem cell differentiation remains to be explored. We took advantage of genetic tools for mtDNA in Drosophila to disrupt mitochondrial OXPHOS in the intestinal stem cells (ISCs). We generated ISCs carrying homoplasmic lethal mtDNA mutation and monitored behaviors of these stem cells and the progenitor cells, enteroblasts (EBs), derived from them. We found that ISCs carrying dysfunctional mitochondria divided much slower to nearly quiescent. Very few progenitors derived from these stem cells failed to differentiate into enterocytes (ECs) or enteroendocrine cells (EEs). Further studies reveal that pathologically elevated FOXO and Notch signaling pathways in the OXPHOS defective ISCs are response for the obstructions of their specification to ECs and EEs, respectively. FOXO pathway negatively regulates ISCs to ECs differentiation, while notch signaling negatively regulates to EE fate. Our results demonstrate that mitochondrial OXPHOS is essential for intestinal stem cells proliferation and differentiation, also suggested that the maintenance of ISC homeostasis is achieved by the complex coordination of mito-nuclear communication that controlling the signaling from inside of organelle to cell surface. Project 5. PPR domain in mitochondrial RNA polymerase is a ribonuclease and essential for priming mitochondrial DNA replication. Mitochondria genome is a compact, circular, double-stranded DNA and its maintenance depends on the dedicated mitochondrial DNA (mtDNA) replication machinery. A number of mitochondrial diseases are characterized by defects in mtDNA replication and a detailed comprehension of the process may help us to understand the pathogenic mechanisms. We here identified that the pentatricopeptide repeat (PPR) domain in mitochondrial RNA polymerase (mtRNApol) possesses RNase activity and is essential for primer synthesis of mtDNA replication. Bacterial protein expression of PPR domain hydrolyzes RNA substrates in a 3-5 manner and requires divalent metal ions for its activity. We further showed that a point mutation from glutamic acid to proline in PPR domain causes loss of RNase function. The RNase-negative point mutation in mtRNApol fails to synthesize RNA primers for mtDNA replication but retains the RNA polymerase function. We also demonstrated that the RNase activity of PPR domain in mtRNApol is highly conserved between drosophila and human. Our work defines a novel function for PPR domain and its importance in mtDNA replication.